Modified ribozymes

ABSTRACT

The present invention refers to an RNA molecule with catalytic activity comprising at least one modified nucleoside, wherein the hydroxy group at the 2′-position of the ribose sugar is replaced by a modifier group, selected from halo, sulfhydryl, azido, amino, monosubstituted amino and disubstituted amino groups, a process for the preparation of modified RNA molecules and the use of modified RNA molecules as therapeutic agents and biocatalysts.

This is a continuation division of application Ser. No. 07/965,411,filed Aug. 9, 1993 now U.S. Pat. No. 5,622,965 which is 371PCT/EP91/01811 filed Sep. 23, 1991.

SPECIFICATION

Certain naturally occuring ribonucleic acids (RNAs) are subject toself-cleavage. The first reported example is the cleavage of theribosomal RNA precursor of the protozoan Tetrahymena (for a review seeCech, Ann.Rev.Biochem. 59 (1990), 543-568) which requires guanosine ascofactor. A number of examples of RNA cleavage have been subsequentlydiscovered in viroid, virusoid and satellite RNAs (for reviews seeSheldon et al. in Nucleic Acids and Molecular Biology (1990) Vol. 4, pg.227-242, ed. F. Eckstein and D. M. J. Lilley, Springer Verlag BerlinHeidelberg; Symons, TIBS 14 (1989), 445-450). These cleavages involvesite-specific breakage of a phosphodiester bond in the presence of adivalent cation such as Mg²⁺, generating a 5′-hydroxyl and a 2′,3′,-cyclic phosphodiester terminus. Sequence analysis around the site ofself-cleavage of several of such RNAs has led to the identification of acommon structural feature essential for cleavage which was named a“hammerhead” structure (Hutchins et al., Nucleic Acids Res. 14 (1986)3627-3640). This structure consists of three helices and 13 conservednucleotides (framed in below scheme) which form a three dimensionalstructure amenable to cleavage at one particular position. Theself-catalyzed cleavage is normally an intramolecular process, i.e. asingle RNA molecule contains all the functions necessary for cleavage.However, Uhlenbeck (Nature 328 (1987), 596-600) nas demonstrated thatthis hammerhead structure does not have to be embodied in one strand butcan be made up of two strands. These two strands combine to form thehammerhead structure which leads to phosphodiester bond cleavage(indicated by an arrow) in one of the strands (strand S) whereas theother (strand E) remains unaltered and can participate in many cleavagereactions. This strand meets the definitions of an enzyme and is calleda ribozyme. Whereas the framed sequences (below scheme) are conservedthe others may vary provided that the structure of base paired and thesingle stranded regions remains intact.

Strand E is SEQ ID NO: 5 and strand S is SEQ ID NO: 8.

The cleavage reaction after the trinucleotide GUC has been studied indetail (Ruffner et al., Gene 82 (1989), 31-41; Fedor and Uhlenbeck,Proc.Natl.Acad.Sci. USA 87 (1990), 1668-1672). Ribozymes with newspecificities have also been constructed (Haseloff and Gerlach, Nature334 (1988), 585-591) indicating that cleavage can for example also takeplace after the sequences GUA, GUU, CUC, AUC and UUC.

Further examples for RNA enzymes are the hairpin RNA (Hampel et al.,Nucleic Acids Res. 18 (1990), 299-304), as well as RNA containingproteins such as the telomerase (Greider and Blackburn, Nature 337(1989), 331-337) and the RNase P (Baer et al., in Nucleic Acids andMolecular Biology (1988), Vol. 3, pp. 231-250, ed. F. Eckstein and D. M.J. Lilley, Springer Verlag, Berlin/Heidelberg).

Ribozymes are potentially of interest for use as therapeutic agents (forreview see Rossi and Sarver, TIBTECH 8 (1990), 179-183). A possiblestrategy would be to destroy an RNA necessary for the expression of bothforeign genes such as viral genes and particular endogenous genes. Thisrequires the construction of a RNA molecule which is able to form ahammerhead or a hairpin structure with the target RNA and to cleave thisat a predetermined position. A first application to the inhibition ofthe HIV-1 virus by this strategy has been reported (Sarver et al.,Science 247 (1990), 1222-1224). Other examples of the action of targetedhammerhead ribozymes in vivo are Cammeron and Jennings(Proc.Natl.Acad.Sci. USA 86 (1986), 9139-9143) and in vitro Cotten etal. (Mol.Cell.Biol. 9 (1989), 4479-4487).

Further, other useful catalytic properties of ribozymes are known, e.g.dephosphorylase and nucleotidyl transferase activities (see PatentApplication WO88/04300). Therein RNA enzymes are disclosed which arecapable of dephosphorylating oligonucleotide substrates with highsequence specifity, which distinguishes them from known protein enzymes.RNA molecules also can act as RNA polymerases, differing from proteinenzymes in that they use an internal rather than an external template.Thus, various heteropolymers can be constructed by variant RNA enzymeforms. This enables the formation for example of messenger RNA moleculesfor particular proteins or peptides. Furthermore, Herschlag and Cech(Nature 344, (1990), 405-409) describe an RNA enzyme with DNaseacitivity.

To be useful as a therapeutic agent the RNA enzyme has to be introducedinto target cells. There are a priori two methods for delivery of theribozyme into the target cells:

-   (a) exogenous delivery of a preformed synthetic RNA;-   (b) endogenous transcription of a ribozyme-coding gene located on a    plasmid.

A great disadvantage of method (a) resides in the very low stability ofRNA molecules under physiological conditions due to their fastdegradation by a variety of ribonuclease enzymes present in the livingcell. The disadvantages of method (b) result from the great difficultiesof specifically and stably inserting a ribozyme-coding gene into thecells of higher organisms. Furthermore, the problem of degradation alsooccurs with in vivo synthesized RNA molecules.

Therefore the problem underlying the present invention was to provideRNA molecules comprising both catalytic activities and enhancedstability against chemical and enzymatical degradation, which can beemployed as therapeutical agents or as biocatalysts in biochemical orbiotechnological processes.

It was however known from a recent paper by Perreault et al. (Nature 344(1990), 565-567) that certain modifications of the RNA enzyme, e.g. theincorporation of 2′-deoxyribo-nucleotides at a few positions of aribozyme lead to a great impairment of the catalytic activity.

It was now surprisingly found that certain chemical modifications at the2′-position of the ribose sugar which enhance the stability of an RNAmolecule do not considerably affect and/or abolish the catalyticproperties of ribozymes.

Therefore it is an object of the present invention to provide an RNAmolecule with catalytic activity comprising at least one modifiednucleoside, wherein the hydroxy group at the 2′-position of the ribosesugar is replaced by a modifier group, selected from halo, sulfhydryl,azido, amino, mono-substituted amino and disubstituted amino groups.

The catalytic activity of an RNA molecule according to the presentinvention comprises advantageously at least one of the group consistingof nucleotidyl transferase, dephosphorylase, deoxyribonuclease and sequnce specific endoribonuclease activities. Preferably the catalyticactivity comprises a sequence specific endoribonuclease activity. Morepreferably the RNA is a hammerhead ribozyme as described above.Especially preferred is that the ribozyme can combine with another RNAstrand to form a hammerhead structure consisting of two strands, whereinthe modified RNA strand is the E strand as described above.

Although a hammerhead ribozyme is especially preferred, other RNAenzymes are encompassed also by the present invention, e.g. theTetrahymena ribozyme (Cech, Ann.Rev.Biochem. 59 (1990), 543-568) innaturally occuring form or a shortened form thereof (Zang et al.,Biochemistry 27 (1988), 8924-8931), and especially the Hairpin RNA(Hampel et al., Nucleic Acids Res. 18 (1990) 299-304) or RNA containingproteins such as the RNase P (Baer et al., in Nucleic Acids & MolecularBiology (1988), Vol. 3, pp 231-250, ed. F. Eckstein and D. M. J. Lilley,Springer Verlag Heidelberg), the telomerase (Greider and Blackburn,Nature 337 (1989), 331-337).

The incorporation of a modifier group at the 2′-position of the ribosesugar appears also to be particularly useful for RNA with new functionseither derived at by a procedure that depends on alternate cycles ofselection (Tuerk and Gold, Science 249 (1990), 505-510; Ellington andSzostak, Nature 346 (1990), 818-822) or any other method.

The modifier group replacing the hydroxy group at the 2′-position of theribose sugar is selected from halo, sulfhydryl, azido, amino,monosubstituted amino, and disubstituted amino groups. The halo groupcan be a fluoro, chloro, bromo or iodo group, wherein the fluoro groupis preferred. The substituents of the substituted amino group arepreferably C₁-C₃ alkyl and or hydroxyalkyl groups. Most preferably themodifier group is a halo or an unsubstituted amino group.

The incorporation of a modifier group at the 2′-position of the ribosesugar significantly increases the RNA stability against enzymaticcleavage. It was confirmed that 2′-deoxy-2′-fluorouridine and2′-deoxy-2′-aminouridine incorporated at specific positions of aribozyme prevented cleavage at these positions by RNase A (see FIG.3+4). This enzyme cleaves at the 3′-position of pyrimidine nucleosidesand requires the presence of the 2′-hydroxyl group (Uchida and Egami(1971), in The Enzymes Vol. IV, 3rd ed. (Ed. P. D. Boyer), AcademicPress, pp. 205-250). Furthermore, results obtained with polynucleotidesshow that the presence of the 2′-amino function also slows downdegradation by unspecific nucleases such as snake venomphosphodiesterase (Hobbs et al., Biochemistry 12 (1973), 5138-5145). Thepresence of a 2′-halogroup also inhibits nucleases such as DNase I(Hobbs et al., Biochemistry 11 (1972), 4336-4344). Results withpolynucleotides also show that the presence of a halogen at the2′-position of a nucleotide protects against the action of human serumnucleases (Black et al., Virology 48 (1972) 537-545). Thus, protectionby incorporation of a modified ribose sugar according to the presentinvention will be rather general and not be restricted to RNases whichdepend on the presence of the 2′-hydroxyl group.

In a ribonucleic acid the ribose sugar is linked to a nucleotide basevia a N-glycosidic bond. The nucleotide base, which is attached to themodified ribose sugar in an RNA molecule of the present invention isselected from the group consisting of bases naturally occuring in RNAand substituted bases. Preferably the modified ribose is attached toadenine, guanine, cytosine and/or uracil, which are the natural bases inRNA. The modified ribose, however, can also be attached to substitutedbases, preferably selected from the group consisting of xanthine,hypoxanthine, 2,6-diamino purine, 2-hydroxy-6-mercaptopurine and purinebases substituted at the 6-position with sulfur or pyrimidine basessubstituted at the 5-position with halo or C₁-C₅ alkyl groups,especially bromo or methyl groups. Most preferably the nucleotide baseattached to the modified ribose sugar is uracil.

The modified nucleosides which are incorporated into a RNA molecule areeither previously described compounds or compounds which can be preparedin analogy to known compounds. The mostly preferred fluoro and aminoanalogs of ribonucleosides have been described previously,2′-deoxy-2′-fluorocytidine (Doerr & Fox, J.Org.Chem. 32 (1967), 1462;Mengel & Guschlbauer, Ang.Chem. 90 (1978), 557-558);2′-deoxy-2′-fluoroadenosine (Ikehara & Miki, Chem.Pharm.Bull. 26 (1978),2449-2453), 2′-deoxy-2′-fluorouridine (Codington et al., J.Org.Chem. 29(1964), 558-564), 2′-deoxy-2′-aminouridine (Verheyden et al.,J.Org.Chem. 36 (1971), 250) and 2′-deoxy-2-aminocytidine (Verheyden etal. (1971) supra). For the synthesis of some of these compounds morerecent synthetic procedures can be employed. The2′-deoxy-2′-fluorocytidine can be prepared from2′-deoxy-2′-fluorouridine by the method of Sung (J.Org.Chem. 47 (1982),3623-3628). The same method can be used for the transformation of2′-deoxy-2′-azidouridine to 2′-deoxy-2′-azidocytidine (Verheyden et al.(1971), supra). The latter can be reduced to 2′-deoxy-2′-aminocytidineby the method of Mungall et al. (J.Org.Chem. 40 (1975), 1659).

The synthesis of the 2′-deoxy-2′-fluoronucleoside 5′-triphosphates canbe carried out either according to Ludwig (Acta Biochim. et Biophys.Acad.Sci.Hung. 16 (1981), 131-133) or Ludwig and Eckstein (J.Org.Chem.54 (1989), 631-635). The 2′-deoxy-2′-aminouridine and -cytidine5′-triphosphates can be prepared as described for the diphosphates byHobbs et al. (Biochemistry 12 (1973), 5138-5145) with the modificationthat pyrophosphate is employed instead of phosphate. The2′-deoxy-2′-fluoronucleoside 3′-phosphoramidites for automatedoligonucleotide synthesis can be prepared by the method of Sinha et al.(Nucleic Acids Res. 12 (1984), 4539-4557). For the synthesis of thecorresponding 2′-amino derivatives, the amino group can be protected bytrifluoroacetylation according to Imazawa and Eckstein (J.Org.Chem. 44(1979), 2039-2041).

An RNA according to the present invention comprises at least onemodified nucleoside, wherein the hydroxy group at the 2′-position ofribose is replaced by a modifier group. A preferred embodiment of thepresent invention is an RNA molecule wherein all nucleosides of one kind(i.e. adenosine or quanosine or cytidine or uridine) contain modifiedsugars, while the remaining three nucleosides contain unmodified sugars.More preferably the modified nucleoside is pyrimidine nucleoside, i.e.cytidine or uridine or a substituted derivative thereof. Most preferablythe modified sugar is 2′-fluoro ribose or 2′-amino ribose. Examples forthis embodiment are the hammerhead ribozymes E2 and E3, which werederived from a hammerhead ribozyme E1 described by Fedor and Uhlenbeck(Proc.Natl.Acad.Sci. USA 87 (1990), 1668-1672). In E2 all uridineresidues are replaced by 2′-deoxy-2′-fluoro-uridine and in E3 alluridine residues are replaced by 2′-deoxy-2′-aminouridine residues. Theribozymes E2 and E3 show a ribonuclease activity which is comparable tothat of the unmodified RNA molecule E1.

In a further preferred embodiment of the present invention allnucleosides of two different kinds contain modified sugars, while theremaining two nucleosides contain unmodified sugars. More preferably allpyrimidine nucleosides, i.e. cytidine and uridine (including substitutedpyrimidine bases) contain modified sugars, most preferably 2′-fluoro or2′-amino ribose derivatives.

Still a further embodiment of the present invention is an RNA moleculecomprising a modification pattern (i.e. which nucleosides are modifiedand which are unmodified) which is designated as a so-called “selectivemodification pattern”. An RNA comprising selective modification patternis a molecule wherein nucleosides at specifically selected locations canbe modified while nucleosides at other specifically selected locationscan be unmodified. For instance, nucleotides which are known to behypersensitive sites for ribonucleases (e.g. due to the secondarystructure of the RNA molecule) should be modified to achieve an extendedlife time of the RNA molecule. An example for aribonuclease-hypersensitive site is provided at position 21 of ribozymeE1. As shown in FIG. 3 the RNA molecule is cleaved at this position byRNase A with very high intensity.

Still a further embodiment of the present invention is a RNA moleculeadditionally comprising at least one modified internucleotidephosphodiester linkage. Examples for suitable modified phosphodiesterlinkages are methyl phosphonate groups or phosphorothioate groups, thelatter being especially preferred. Preferably at least the 5′-terminalphosphodiester linkage and/or the 3′-terminal phosphodiester linkage ofthe RNA molecule is modified. More preferably the 5′-terminalphosphodiester linkage and the last three 3′-terminal phosphodiesterlinkages are modified.

It was found, that the presence of modified internucleotidic linkagesalone was not sufficient to provide increased stability againstdegradation. However, the combined presence of 2′-modified ribose sugarstogether with modified internucleotidic linkages showed an additivestability enhancing effect. A more than fiftyfold increase in stabilityconfered by both modifications outweighs the decreased efficiency incleavage compared to a unmodified ribozyme.

The synthesis of RNA molecules having modified inter-nucleotidiclinkages is preferably accomplished by means of chemical synthesis asdescribed below.

A further object of the present invention is a process for the synthetisof an RNA molecule with catalytic activity, comprising:

incorporating into an RNA chain at least one modified nucleoside,wherein the hydroxy group at the 2′-position of the ribose sugar isreplaced by a modifier group, selected from halo, sulfhydryl, azido,amino, monosubstituted amino and disubstituted amino groups.

Preferably the modifier group is a halo (i.e. a fluoro, chloro, bromo oriodo group) or an amino group, more preferably a fluoro or anunsubstituted amino group. It should be noted, that the process of thepresent invention also comprises the synthesis of an RNA moleculewherein nucleotides with at least two different modifier groups (e.g.fluoro and amino groups) are incorporated.

There are preferably two approaches for the incorporation of thesemodified nucleotides into RNA. One is by automated chemical synthesis ofRNA molecules which can be carried out on solid support or in solution,preferably with the respective phosphoramidites or H-phosphonates asnucleotide precursors, the other involves enzymatic incorporation bytranscription of appropriate nucleic acid, preferably DNA templates witha nucleic acid polymerase using the 2′-modified nucleoside5′-triphosphates. By means of automated chemical synthesis RNA moleculescomprising modified internucleotidic linkages may be prepared byincorporating the corresponding chemically modified nucleotideprecursors such as the methyl phosphonate derivatives into the RNAchain. For the incorporation of phosphorothioate linkages the standardphosphoramidite derivatives are used as nucleotide precursors. After thecoupling of the precursor to the RNA chain has taken place thesubsequent oxidation step, however, is not performed with iodine, as inthe case of non-modified linkages, but with sulfur or a sulfuratingagent, whereby the phosphorothioate group is obtained.

The chemical synthesis of modified RNA molecules is carried out inanalogy to that of unmodified RNA or DNA molecules, which is known inthe art. More specifically the RNA synthesis is carried out by chemicalsynthesis on solid support involving the stepwise addition of therespective nucleotide precursors. After having synthesized an RNAproduct of the desired length, the RNA is removed from the solid supportby conventional means and purified, preferably by gel electrophoresis.Alternatively the chemical RNA synthesis can also be carried out by anyother known technique without using a solid support. E.g. the RNA can besynthesized in a soluble form and subsequently purified by means knownin the art.

When the 2′-amino modifier group is incorporated into the RNA chain ithas to be protected before the phosphitylation reaction (i.e. thepreparation of the nucleotide precursor) and for subsequent use in thecoupling reactions. For this purpose the trifluoroacetyl group ispreferably used as a protecting group, because it is stable during thecycles of synthesis on the nucleic acid synthesizer and is removableunder the conventional treatment with ammonia.

Alternatively the synthesis of the RNA chain can be carried out bytranscription from a nucleic acid template by an appropriate nucleicacid polymerase. Preferably the template is a DNA template and thenucleic acid polymerase is a DNA dependent RNA polymerase. Morepreferably the DNA dependent RNA polymerase is selected from the groupconsisting of T7, T3 and SP6 polymerases, which are highly processivebacteriophage RNA polymerases. Among these polymerases the T7 RNApolymerase is most preferred. The DNA template for the synthesis of amodified RNA molecule according to the present invention is preferablyconstructed by inserting a synthetic DNA fragment coding for the desiredRNA sequence into an appropriate site of a plasmid, wherein said plasmidcomprises a promoter for the respective RNA polymerase and said site islocated at such a position of the plasmid, so that the synthetic DNAfragment can be transcribed from said promoter. The transcriptionreaction is advantageously carried out as a run off transcription.Alternatively, synthetic DNA fragments may serve as transcriptiontemplates without a plasmid portion. Those fragments, however, shouldcontain a transcription start signal, which allows an effective RNAsynthesis.

The polymerisation of 2′-deoxy-2′-halo nucleotides, e.g.2′-deoxy-2′-fluorouridine, -cytidine, -adenosine, -guanosine and therespective chloro compounds, is preferably carried out by T7 polymerasein the presence of Mn²⁺ ions as cofactor. Alternatively, thepolymerisation of 2′-aminonucleotides, e.g., 2′-deoxy-2′-aminouridine2′-deoxy-2′-aminocytidine, 2′-deoxy-2′-aminoadenosine, and2′-deoxy-2′-aminoguanosine, is preferably carried out in the presence ofMg²⁺ ions as cofactor.

From the experimental data of the following examples it is evident thatthe presence of 2′-deoxy-2′-fluorouridine and 2′-deoxy-2′-aminouridinein a hammerhead ribozyme do not abolish catalytic activity. This isqualitatively shown in FIG. 3 for the presence of the 2′-fluorouridinein the substrate part and quantitatively in Table 1 for various otherenzyme/substrate pairs. It is true that all the modifications resultedin an increase in the K_(m)-value which was most pronounced for theamino substitution. However, this perturbation of the active structurelies well within the range of Km variation observed for hammerheadsystems with different base composition (Fedor & Uhlenbeck, supra). Inaddition, very surprisingly the incorporation of a single2′-aminouridine immediately 5′ of the site of cleavage in the substrateincreased the kcat markedly (table 1), so that it is conceivable toproduce ribozymes of enhanced activity by the selective introduction of2′-modified nucleosides at specific sites. These results definitely showthat there is no requirement for the presence of 2-hydroxyl groupsthroughout the enzyme part of the hammerhead structure for catalyticactivity but that the modifications according to the present inventionare tolerated at least in certain positions. In contrast, theincorporation of only 15% 2′-deoxynucleotides into a hammerhead ribozymeis reported to decrease the catalytic efficiency by two orders ofmagnitude, while not affecting the K_(m) (Perreault et al. (1990),supra). Since the rate of cleavage is determined by the angle of attackof the 2′-hydroxyl on the phosphorus at the site of cleavage, it isgreatly influenced by the overall structure of the hammerhead system.Thus, the observed influence of 2′-modifications on the rate supportsthe notion that the 2′-fluoro analogs adopt a structure more similar tothat of ribonucleotides than that of deoxyribonucleotides. Thisapparently also holds for the amino analogs. The other 2′-modifiednucleosides according to the present invention exhibit similar catalyticactivity.

A still further object of the present invention is the use of RNAmolecules with catalytic activity comprising at least one modifiednucleotide, as therapeutic agents, especially for the specific cleavageof viral or other foreign genetic material or transcripts from viral orother foreign genetic material, or as biocatalyst in biochemical orbiotechnological processes. For these purposes the RNA molecules of thepresent invention seem to be more suitable than their unmodifiedanalogs, because of their increased stability against chemical and/orenzymatical cleavage.

The present invention shall be further illustrated by the followingexamples in combination with FIGS. 1-7. These examples however are notintended to narrow the scope of the present invention.

FIGS. 1 a and 1B shows autoradiographs of T7 RNA polymerase run offtranscripts of the plasmid pUCRS after PAGE.

FIG. 2 shows an autoradiograph of T7 RNA polymerase run off transcriptsof the plasmid pUCRE containing 2′-aminouridine after PAGE.

FIG. 3 shows an autoradiograph of partial Ribonuclease A cleavage of5′-labeled run off transcripts E1 and E2 separated by PAGE.

FIG. 4 shows an autoradiograph of the total degradation of S1 and S2 byRNase A.

FIG. 5 shows an autoradiograph of the cleavage of 2′-fluorouridine and³²P-AMP containing substrate S3 by ribozyme E1.

FIG. 6 shows an Eadie-Hofstee plot of the ribozyme reaction of E2 withS1.

FIG. 7 shows an Lineweaver-Burk plot of the ribozyme reaction of E3 withS1.

FIG. 8 shows the organisation of the HIV-1 sequence cloned into pOTH33.

FIG. 9 shows the nucleotide sequence of the ribozyme RE115.

EXAMPLES Example 1

Preparation of Oligoribonucleotides

Automated synthesis of oligoribonucleotides: Automatedoligoribonucleotide synthesis was carried out with an Applied Biosystems380B DNA Synthesizer on a 1 μmol scale using the monomericribonucleotide phosphoramidites supplied by Milligen/Biosearch. Controlpore glass columns with the ribonucleoside coupled to it were eitherfrom Milligen/Bio-search or Peninsula. The oligomers were worked upaccording to the specifications of the supplier of the ribonucleotidephosphoramidites (Milligen/Biosarch). After removal of the protectinggroups the oligoribonucleotides were concentrated by spin dialysis onAmicon filter membranes centricon 10 and ethanol precipitated. The driedpellets were taken up in 50 μl water and subjected to PAGE. Bands werevisualized by UV shadowing, cut out and the RNA was isolated by elutingat 37° C. overnight in buffer (0.25 M ammonium acetate, 10 mM TRIS/HClpH 8.0, 1 mM EDTA) (Fedor & Uhlenbeck, PNAS USA 87 (1990), 1668-1672).Concentrations were determined using the extinction coefficient pernucleotide of 6600 M⁻¹ cm⁻¹ given in the literature (Fedor & Uhlenbeck1990). Aqueous solutions of the oligoribonucleotides were stored at −20°C.

Construction of plasmids containing templates for run off transcription:

The following oligodeoxynucleotides were synthesized for the plasmidconstruction by the phosphoramidite method with an Applied Biosystems380B DNA synthesizer:

RS2-T,5′-d(GATATCCTAGACTCCCTATAGTGAGTCGTATTA-3 (SEQ ID NO: 1),RS2-C,5′-d(TAATACGACTCACTATAGGGAGTCAGGATATCTGCA-3′(SEQ ID NO:2);RE1-T,5′-d(GGAGTTTCGGCCTAACGGCCTCATCAGAGGACCCTATAGTGAGTCGTATTA)-3′(SEQID NO:3); andRE2-C,5′-d(TAATACGACTCACTATAGGGTCCTCTGATGAGGCCGTTAGGCCGAAACTCCTGCA)-3′(SEQID NO: 4)

Preparation of ribozyme pUCRS and pUCRE16 clones:

The commercially available plasmid pUC19 was cleaved in a one stepreaction using the restriciton enzymes Iso-SacI and PstI. The DNA wasthen purified by 2% agarose gel electrophoresis followed byelectroelution using a Centricon 30 and the centroelution apparatussupplied by Amicon. The oligonucleotide primer pairs, RE1-T and RE2-C(ribozyme enzyme), or RS2-T and RS2-C (ribozyme substrate) werephosphorylated as previously described (Taylor et al., Nucleic AcidsRes. 13 (1985), 8749-8764). These oligonucleotide pairs were used forcloning of the T7 promoter along with either the DNA sequence for theribozyme yielding pUCRE16 or the ribozyme substrate yielding pUCRSaccording to the procedure of King & Blakesley (Focus 8 (1986), 1-3).After transformation of competent cells (Olsen & Eckstein, PNAS USA 87(1990), 1451-1456) white colonies were screened for the presence of asecond AvaII site in the case of the pUCRE16 or a unique EcoRV site forpUCRS. The sequence of the purified double-stranded DNA from each clonewas determined by the procedure of Olsen and Eckstein (Nucleic AcidsRes. 17 (1989), 9613-9620).T7 RNA polymerase run off transcripts:T7 RNA polymerase run off transcripts were synthesized on a 150 μl to500 μl scale by adapting the procedure given by Milligan and Uhlenbeck(Meth. in Enzymology 180A (1989), 51-62). Transcription reactions wererun in 40 mM TRIS pH 8.0, 1 mM spermidine, 5 mM DTT, 0.01% Triton x-100,20 mM MgCl₂, 2.5 mM nucleotides, 200 μM DNA template, 0.2 U/μl humanplacental RNase inhibitor, and 100 U/μl T7 RNA polymerase. When2′-deoxy-2′-fluoronucleoside triphosphates were employed, the MgCl₂ wasreplaced by 20 mM MnCl₂. Reactions were run at 37° C. for 3 hours.Transcripts were purified by PAGE as described above. Aqueous solutionsof the oligoribonucleotides were stored at −20° C.

FIG. 1 shows autoradiographs of T7 RNA polymerase run off transcriptionsof pUCRS after PAGE. A: The transcription was performed on a 150 μLscale in the presence of 20 mM MgCl₂ and 2.5 mM each of the fournucleoside triphosphates at 37° C. for 3 h. The reaction mixture wasdephosphorylated with alkaline phosphatase and 5′-³²P-labeled byreaction with polynucleotide kinase and [δ-³²P]-ATP. The labeledtranscription mixture was subjected to PAGE. B: The transcription wasperformed on a 150 μL scale at 37° C. for 3 h in the presence of 20 mMMnCl₂, 0.5 mM ATP, 25 μCi [α-³²P]-ATP, 2.5 mM CTP and GTP, and 2.5 mM2′-fluorouridine triphosphate. The transcription mixture was directlysubjected to PAGE. The asterisks mark ³²P-labeled phosphates. ‘N’denotes any nucleotide added by T7 RNA polymerase beyond the full lengthof the template DNA (c.f. Milligan and Uhlenbeck, Meth.in Enzymology180A (1989), 51-62).

FIG. 2 shows an autoradiograph of T7 RNA polymerase run off transcriptsof pUCRE 16 containing 2′-aminouridine after PAGE. Lane 1:2′-aminouridine containing 34-mer marker E3, synthesized chemically.Lane 2: The transcription was performed on a 150 μl scale at 37° C. for3 h in the presence of 20 mM MgCl₂, 60 μCi [α-³²P]ATP, 1 mM CTP and GTP,and 1 mM 2′-aminouridine triphosphate. The transcription mixture wasdirectly applied PAGE.

Preparation of oligoribonucleotides: The following oligoribonucleotideswere prepared

-   a.) by run off transcription (sequences given without the    5′-triphosphate):-   E1. 5′-GGGUCCUCUGAUGAGGCCGUUAGGCCGAAACUCC-3′(SEQ ID NO:5);-   E2,5′-GGG(2′-FU)CC(2′-FU)C(2′-FU)GA(2′-FU)GAGGCCG(2′-FU)(2′-FU)AGGCCGAAAC(2′-FU)CC-3′    (SEQ ID NO:6) and-   E3,5′-GGG(2′-NH₂U)CC(2′-Nh₂U)C(2′-NH₂U)GA(2′-NH₂U)GAGGCCG(2′-NH₂U)(2′-NH₂U)AGGCCGAAAC(2′-NH₂U)CC-3′    (SEQ ID NO:10).-   b.) by chemical synthesis: The oligoribonucleotides E1, E2, E3, S1    and S2, 5′-GGGAG(2′-NH₂U)CAGGAU-3′ (SEQ ID NO:11).

5′-³²P-labeling of oligoribonucleotides:

Oligoribonucleotides obtained from run off transcriptions weredephosphorylated by treatment with RNAse free bovine alkalinephosphatase, purified by Quiagen tip-20 columns according to theprotocol given by the manufaturer (Diagen Inc.) and treated with T4polynucleotide kinase and δ-³²P-ATP. Labeled oligoribonucleotides werepurified by PAGE.

Example 2

Digestion of Oligoribonucleotides with RNase A

Partial digestion of oligoribonucleotides with RNase A: Theoligoribonucleotides E1 and E2 were subjected to RNase A digestion after5′-³²P labeling according to the procedure of Donis-Keller et al.(Nucleic Acids Res. 4 (1977), 2527-2538) with the following changes.Approximately 25 μmoles of 5′-³²P-labeled RNA was added to 50 μl buffercontaining 7 M urea, 50 mM EDTA, 0.04% bromophenol blue, 0.04% xylenecyanol FF and 0.25 mg/ml tRNA on ice. The RNA was then equally dividedinto 5 labeled tubes, heated to 50° C. for 5 min and then immediatelyplaced on ice. Ribonuclease A, 2 μl (2×10⁻⁴ units), was added to thefirst tube and mixed using the pipette. The enzyme was then successively5 fold diluted into three additional tubes using a new pipette tip aftereach transfer from one tube to the next. The fifth tube was a controlsample to which no enzyme was added. All tubes were then incubated at50° C. for 5 min, placed on ice and analysed by PAGE.

Total degradation of oligoribonucleotides by RNAse A: Theoligoribonucleotides S1 and S2 were digested with RNase A after 5′-³²Plabeling according to the following protocol: The oligomer (8.5 μM in afinal volume of 20 μl) was reacted with 1.25×10⁻³ Units of RNAse A inbuffer containing 50 mM TRIS/HCl pH 7.5 and 10 mM MgCl₂ for 10 min at37° C. Products were analyzed by PAGE.

FIG. 3 shows an autoradiograph of partial Ribonuclease A cleavage of5′-labeled run off transcripts E1 and E2 separated by PAGE. Conditionsas described before. The numbered lanes correspond to 1) no enzymeadded, 2) 2×10⁻⁴ units RNase A, 3) 3×10⁻⁵ units RNase A, 4) 8×10⁻⁶ unitsRNase A, 5) 16×10⁻⁷ units RNase A. Base numbering was facilitated bycounting the bands of a Mn²⁺ mediated cleavage of the unmodifiedtranscript (10 μmoles RNA heated to 90° C. for 3 min in 10 mM MnCl₂).The circled numbers indicate the bands expected from RNase-A susceptiblecleavage positions. Arrows indicate the bands that arise from cleavage3′ to uridine and which are absent in the lanes where 2′-fluorouridinecontaining ribozyme was cleaved.

FIG. 4 shows an autoradiograph of the total degradation of S1 and S2 byRNase A after PAGE. Details of the reaction are as described above. Lane1: total digestion of 12-mer S2; lane 2: total digestion of 12-mer S1;lane 3: cleavage ladder of the 34-mer E1 by reaction with 20 mM MnCl₂ at90° C. for 3 min as a length standard. The product of cleavage of S2 is1 nucleotide longer than that of S1 indicating the presence of2′-aminouridine at position 6.

Example 3

Cleavage of Oligoribonucleotide Substrates by Ribozymes

Determination of cleavage kinetics: The cleavage reactions wereperformed by a procedure adapted from Fedor and Uhlenbeck (1990),supra). Stock solutions of the ribozyme enzyme (typically 20 μL finalvolume, 100 nM final concentration, 50 mM TRIS/HCl, pH 7.5) andsubstrate oligonucleotide (typically 40 μl, 2 μM final concentration)were heated separately at 90° C. for 1 min and cooled to roomtemperature for 15 min prior to the addition of divalent metal ion(MnCl₂ or MgCl₂, 10 mM final concentration). These stocks were incubatedseparately at 25° C. for 15 min prior to initiation of the cleavagereactions. The reactions were started by adding enzyme to substrate (50mM TRIS/HCl, pH 7.5, 20 μl final volume, MgCl₂, 10 mM finalconcentration), with typical concentrations of 10 nM enzyme and between50 and 5000 nM substrate. At set times 10 mM aliquots were transferredinto 10 mM urea stop mix and subjected to PAGE. Autoradiographs wereanalyzed on an LKB ULTROSCAN XL laser densitometer.

In the investigated hammerhead ribozyme system a 12-mer substrateoligonucleotide (designated as S) is cleaved by a 34-mer enzymeoligonucleotide (designated as E) at the 3′-position of cytidine-7 asindicated by the arrow in the structure in the Introduction. Thiscleavage generates a heptamer with a 2′-3′-cyclic phosphate terminus(product 1) and a pentamer with a 5′-hydroxyl terminus (product 2)(Ruffner et al, Gene 82 (1989), 31-41). We observed these types ofcleavage products not only with the oligoribonucleotides E1 and S1, butalso with the 2′-fluorouridine-containing substrate S3 (FIG. 5). Asexpected, the substrate oligonucleotide S4, containing a2′-fluorocytidine at the position of cleavage was not cleaved underidentical conditions. These two reactions contained 2′-fluorouridine inthe substrate oligonucleotide.

However, potentially more interesting for future applications is thequestion whether the presence of this modification in the enzyme part ofthe ribozyme will interfere with its catalytic activity. Thus, thereaction of the 2′-fluorouridine-containing ribozyme E2 with theunmodified substrate S1 was investigated. Indeed, the gel analysisindicated that the substrate was cleaved with similar efficiency as thepair E1 and S1. The catalytic constants of the2′-fluorouridine-containing ribozyme E2 were determined (FIG. 6) andcompared to those of the unmodified ribozyme E1. This comparison revealsthat the second order rate constant for the former (k^(cst)/K_(m)=0.0026nM⁻¹) is one order of magnitude smaller than that of the latter(k_(cat)/K_(m)=0.023 nM⁻¹) (Fedor & Uhlenbeck (1990), supra) (Table 1).This decrease in catalytic efficiency is primarily due to a decrease inthe rate of cleavage, whereas the K_(m) values for both ribozymes isnearly identical. This reduced rate of cleavage, however, lies wellwithin the range of cleavage efficiencies observed for varioushammerhead systems with different base compositions (Fedor & Uhlenbeck(1990), supra). Hammerhead ribozyme reactions can be carried out withMgCl₂ as well as with MnCl₂ as metal ion cofactor, where the half lifeof cleavage is decreased in the presence of the latter cofactor by about10 fold (Uhlenbeck, Nature 328 (1987), 596-609). Such a decrease in thehalf life of the substrate under cleavage conditions upon switching fromMg²⁺ to Mn²⁺ was also observed for the reaction of2′-fluorouridine-containing enzyme E2 with substrate S1. Thus the metalion requirement for the cleavage reaction is not altered by theincorporation of 2′-fluoronucleotide analogs.

The effect of the presence of 2′-aminouridine in the ribozyme was alsoinvestigated. When the 2′-aminouridine containing ribozyme E3 is reactedwith nonmodified substrate S1, the catalytic efficiency drops an orderof magnitude to k_(cat)/K_(m)=0.0015 nM⁻¹. This decrease in efficiencyis clearly due to a higher K_(m), while the k_(cat) remains almostunaltered. Thus, the overall efficiency of the 2′-aminouridine ribozymeis comparable to the one of the 2′-fluorouridine containing ribozyme. Inthe complementary reaction of the nonmodified ribozyme E1 with theselectively 2′-aminouridine modified substrate S2 the catalyticefficiency is increased compared to the above reaction tok_(cat)/K_(m)=0.0063 nM⁻¹. This effect is entirely due to an increase ink_(cat). This trend is even more pronounced in the reaction of the2′-aminouridine containing ribozyme E3 with S2, where the catalyticefficiency is increased to k_(cat)/K_(m)=0.011 nM⁻¹, again mainly due toan increased k_(cat). The kinetic parameters for all of the abovereactions are summarized in Table 1.

TABLE 1 Kinetic constants of 2′-modified nucleotide-containingribozymes.^(a) k_(cat) K_(m) k_(cat)/K_(m) Enzyme Substrate (min⁻¹) (nM)(nM⁻¹ min⁻¹) E1 (nonmod.) S1 (nonmod.) 3.0  140 0.023  E2 (2′-FU) S1(nonmod.) 0.8  300 0.0026 E3 (2′-NH₂ U) S1 (nonmod.) 2.3 1500 0.0015 E3(2′-NH₂ U) S2 (2′-NH₂ U) 19.0  1800 0.011  E1 (nonmod.) S2 (2′-NH₂ U)10.0  1600 0.0063 ^(a)Kinetic constants were determined fromEadie-Hofstee plots of cleavage reactions run with 10 nM ribozyme andwith substrate concentrations ranging from 50 nM to 1200 nM.

Thus, the herein compiled kinetic data shows that while the cleavageefficiency of 2′-fluoro- and 2′-aminouridine modified ribozyme issomewhat reduced, it is still within the range of variations observedfor hammerhead systems of different base composition. It also becomesevident that it is possible to increase the catalytic efficiency byselectively introducing 2′-modifications at specific positions. Whilethe latter effect was demonstrated for the substrateoligoribonucleotide, it is anticipated that a similar influence oncatalysis can be found for selective modifications in the enzyme.

FIG. 5 shows an autoradiograph of the cleavage of 2′-fluorouridine and³²P-AMP-containing substrate S3 by ribozyme E1. The cleavage reactionwas performed in the presence of 10 mM MgCl₂ in 50 mM TRIS/HCl, pH 7.5on a 40 μl scale at 25° C. The concentration of E1 and S3 was 2.5 μM and7.5 μM, respectively. All other details are as described above (c.f.Determination of Cleavage Kinetics). At the indicated times 10 μlaliquots were transferred into 10 μl water and 10 μl urea stop mix priorto PAGE. Lane 1: reaction after 0.5 min; lane 2: reaction after 15 min;lane 3: reaction after 30 min. The asterisks mark ³²P-labeledphosphates.

FIG. 6 shows an Eadie-Hofstee plot of the ribozyme reaction of E2 withS1. The cleavage reaction were performed on a 20 μl scale in thepresence of 10 mM MgCl₂ with a 10 nM concentration of E2 andconcentrations of S1 of 50 nM, 100 M, 200 nM, 400 nM, 500 nM, and 700nM. After 7 min 10 μl aliquots were transferred into 10 μl water and 10μl urea stop mix prior to PAGE. It was established previously that thesetime points fall within the linear range of initial velocities. Theautoradiographs were evaluated by integration of their optical densityon a laser densitometer.

FIG. 7 shows an Lineweaver-Burk plot of the ribozyme reaction of E3 withS1. The cleavage reactions were performed on a 20 μl scale in thepresence of 10 nM MgCl₂ with a 10 nM concentration of E3 andconcentrations of S1 of 50 nM, 100 nM, 200 nM, 400 nM, 500 nM and 700nM. All other details are as in FIG. 6.

Example 4

Cleavage of HIV-1 LTR RNA Using Ribozymes

Plasmid Construction: A plasmid, pOTH33, was constructed by cloning theHIV-1 sequence from position −525 to 386 (according to the sequencenumbering by Ratner et al., Nature 313 (1985), 277-284) into thecommercially available plasmid pSPT19 (Pharmacia). The HIV sequence isunder transcriptional control of a T7 promotor (T7). A diagrammatic viewof the HIV insertion in pOTH33 is given in FIG. 8. The HIV-1 LTR regionconsists of the U3 region, the R region and the U5 region. It is flankedon its 5′-end by the polypurine tract and on tis 3′-end by the primerbinding site (PBS), the leader sequence and a part of the gag gene. Thearrows at position −525 and 386 indicate the restriction sites used forthe construction of pOTE33. The arrow at position 115 shows the site forribozyme mediated cleavage.

RNA of HIV-1 from position −525 to 386 comprising the long terminalrepeat sequence from nucleotide −453 to 182 was obtained by run-offtranscription of EcoRI cleaved plasmid pOTH33 (100 ng/μl DNA template,10 mM DTT, 500 μM of each rNTP, 50 mM Tris-Cl pH 7.5, 2 mM spermidine, 6mM MgCl₂,2 μci/μl [α-³²P]-ATP, 50 U/μl RNase inhibitor and 15 U/μl T7RNA polymerase, 2 h at 4° C.) and subsequent incubation of the reactionmix with DNaseI (1 U/μl, 10 min at 37° C.) (RNase free) andphenol-chloroform extraction. The obtained RNA was designated as LTRRNA.

Position 115 of the HIV-1 LTR RNA containing the potential cleavage siteGUC was chosen as a target for ribozyme catalyzed cleavage. Hammerheadribozymes targeted against this site were chemically synthesized. Thenucleotide sequence of the unmodified hammerhead enzyme RE115 is givenin FIG. 9.

Cleavage Kinetics with LTR RNA: k_(cat)/K_(m) values were determinedunder single turnover conditions. Ribozymes were preincubated at 75° C.for 1 min in the presence of 50 mM Tris-Cl pH 7.5 followed by 5 min ofincubation at 37° C. MgCl₂ was added to a final concentration of 10 mMand the solutions were again incubated for 5 min at 37° C. LTR RNA wasdirectly used as an aqueous solution. The reaction mixture (10 μl)contained between 20 nM and 1 μM ribozyme, 50 mM Tris-Cl pH 7.5 and 10mM MgCl₂. The reaction was started by addition of LTR RNA to a finalconcentration of 10 nM. After 1 hour at 37° C. the reaction was stoppedby addition of 10 μl stop mix and analysed by 4% PAGE (40 cm long, 8 Murea). After 1 h electrophoresis at 50 W followed by autoradiography thefraction of noncleaved LTR RNA was determined by laser scanningdensitometry. k_(cat)/K_(m) values were obtained by plotting theremaining fraction of LTR RNA (Frac S) against the ribozymeconcentration ([RE]) according to the following equation:${k = {\frac{\ln\quad({FracS})}{t} = {\lbrack{Re}\rbrack\frac{k_{cat}}{K_{m}}}}},$where k is the observed reaction rate and t is the reaction time of 1 h.

In order to investigate the influence of chemical modifications on thecatalytic efficiency of a ribozyme several analogs of RE115 containing2′-fluoro or 2′-deoxy substitutions and/or terminal phosphorothioatelinkages were synthesized. Whereas 2′-fluorocytidine substitutions hatno effect on the catalytic efficiency [Table 2, RE115(FC)],2′-fluorouridine substitutions caused a fivefold decrease ofk_(cat)/K_(m) [Table 2, RE115(FU)]. One 5′-terminal phosphorothioategroup in combination with three 3′-terminal phosphorothioate groupsdiminished the catalytic efficiency only negligibly [Table 2, RE115(S)].The same was true for the combination of terminal phosphorothioatelinkages together with 2′-fluorouridine substitutions, where no. furtherdecrease in k_(cat)/K_(m) was observed [Table 2, RE115(FU),S)].Substituting all pyrimidine ribonucleotidse by their 2′-fluoro analogsand introducing the four phosphorothioate linkages decreased thecatalytic efficiency only sevenfold compared to the unmodified ribozyme[Table 2, RE115(FC,FU,S)]. In contrast substitutions of all pyrimidineribonucleotides by their 2′-deoxynucleoside analogs combined withphosphorothioates resulted in a decrease of k_(cat)/K_(m) by a factor of50 [Table 2, RE115(dC,dU,S)]. Thus, RE115(dC, dU,S) is some 7 times lessefficient than RE115(FC,FU,S).

TABLE 2 Influence of chemical modifications on the Cleavage of LTR RNAby RE115 k_(cat)/K_(m), k_(cat)/K_(m), Ribozyme M⁻¹ s⁻¹ relative RE115500 1 RE115(S) 360 0.72 RE115(FC)¹ 490 0.98 RE115(FU)¹  89 0.18RE115(FU,S)¹  59 0.12 RE115(FC,FU,S)¹  69 0.14 RE115(dC,dU,S)²  10 0.020¹Examples of the present invention ²Comparative example

Example 5

Stability of Oligoribonucleotides

The ribozymes of Example 4 were examined for their stability againstnuclease digestion.

Test conditions:

Molt 4 clone 8 cells (kindly supplied by E. Jurkiewicz, DeutschesPrimatenzentrum, Göttingen) grown in medium RMPI 1640 to a cell densityof about 10⁶ cells/ml were centrifuged at 1000 g for 5 min in a HeraeusMinifuge. 5′-³²P-labeled ribozymes were pre-heated for 1 min at 90° C.,chilled on ice, added to the cell supernatant to a final concentrationof 300 nM and incubated at 37° C. Aliquots were taken at the indicatedtime points and analysed by 20% PAGE containing 8 M urea followed byautoradiography.Results:

More than 80% of RE115 was degraded after 2 min incubation in the cellsupernatant as indicated by denaturing PAGE. For RE115(S) similarresults were obtained. However, no degradation of RE115(FC,FU,S) within1 hour was observed. A comparison with the rate of degradation of theunmodified ribozyme indicates that the combination of 2′-modifiedpyrimidine nucleosides and terminal phosphorothioate linkages results inan estimated increase of more than fiftyfold of ribozyme stabilityagainst digestion by nucleases present in T cell supernatant.2′-modified ribozymes without phosphorothiate group show a stabilitywhich is about two times lower than the stability of RE115 (FC,FU,S).

1. A method of cleaving a target RNA molecule comprising the step ofproviding said target RNA molecule with a catalytic RNA molecule underconditions suitable for said catalytic RNA molecule to cleave saidtarget RNA molecule, wherein said catalytic RNA molecule comprises atleast one modified nucleoside, said modified nucleoside comprising amodifier group, selected from the group consisting of halo, sulfhydryl,azido, amino, monsubstituted amino and disubstituted amino groupsreplacing a hydroxy group at the ribose sugar 2′-position.
 2. The methodof claim 1, wherein said modifier group is a halo group.
 3. The methodof claim 1, wherein said modifier group is an amino group.
 4. The methodof claim 1, wherein said modifier group is a monosubstituted aminogroup.
 5. The method of claim 1, wherein said modifier group is adisubstituted amino group.
 6. The method of claim 1, wherein saidmodifier group is an azido group.
 7. The method of any of claims 1-6,wherein said catalytic RNA molecule is a hammerhead ribozyme.
 8. Themethod of any one of claims 1-6, wherein said catalytic RNA molecule isa hairpin RNA.
 9. The method of any one of claims 1-6, wherein saidtarget RNA molecule is a foreign genetic material.
 10. The method ofclaim 7, wherein said target RNA molecule is a foreign genetic material.11. The method of claim 8, wherein said target RNA molecule is a foreigngenetic material.
 12. The method of any of claims 1-6, wherein saidtarget RNA molecule is viral material.
 13. The method of claim 7,wherein said target RNA molecule is viral material.
 14. The method ofclaim 8, wherein said target RNA molecule is viral material.